The GAP in Fruit Development: Determining the Role of RanGAP in Arabidopsis Fruit Development Honors Research Thesis Presented in Partial Fulfillment of the Requirements for graduation “with Honors Research Distinction in Plant Cellular and Molecular Biology” in the undergraduate colleges of The Ohio State University By Bailey Tilford The Ohio State University April 2014 Project Advisor: Professor Iris Meier, Department of Molecular Genetics 1 Abstract: RanGAP is the activating protein for the small GTPase Ran, and is known to be involved in nucleocytoplasmic transport and mitotic cell division across kingdoms. Arabidopsis has two RanGAP proteins, RanGAP1 and RanGAP2, which share 63% amino acid homology. Both proteins contain two functional domains: a localization domain known as the WPP domain, which contains a conserved tryptophan- proline-proline motif necessary for interaction with localization binding partners, and a GTPase activation (GAP) domain, which is responsible for Ran binding and activation. Double null mutants in the genes encoding RanGAP1 and RanGAP2 are lethal at an early stage of development. However, a homozygous mutant combining the RanGAP1 null allele and RanGAP2 knockdown allele, called short silique knockdown (SILK), exhibits a reduced fruit (silique) length phenotype. What role RanGAP plays in fruit development, however, is unknown. In order to determine its function, constructs containing wildtype RanGAP1 or mutant RanGAP1 with one or both functional domains mutated were inserted into SILK mutant plants. Their phenotypes were quantified by measuring fruit length and seed number to determine if the RanGAP1 transgene had rescued the short-fruit phenotype. RanGAP1 transgenes with mutations in the GAP domain did not rescue the SILK phenotype whereas mutants that maintained this function did, regardless of localization. These results indicate that it is the GAP function of RanGAP that is important in fruit development. Seed count, however, varied greatly among individual lines with the same transgene, indicating that seed development may depend on the location of the RanGAP1 insertion into the genome. Further analysis of silique cell types indicate that the length phenotype may be due to defects in the differentiation of the cells of the fruit itself. This work highlights the intersection between cell biological processes and developmental events, and shows the relevance of intracellular events to the understanding of whole-plant processes. Introduction: RanGAP RanGAP (Ran GTP-ase Activating Protein) is the activating protein of the small GTPase Ran: a small Ras superfamily GTP-ase primarily involved in the transport of 2 RNA and protein between the nucleoplasm and the cytoplasm, with homologues in many different species1. This transport role is dependent on a gradient of Ran across the nuclear membrane, with GTP-bound and GDP-bound forms dominant in the nucleoplasm and the cytoplasm, respectively2. The transition between these forms is dependent on the interaction of Ran with its activating protein, RanGAP, and its nucleotide exchange factor, RanGEF2–4. The ability of RanGAP to help Ran to hydrolyze GTP is referred to as its GTPase-activation (GAP) activity 5–7. This interaction occurs via the leucine-rich repeat (LRR) domain of the protein8. All known RanGAP proteins contain an LRR domain, as well as a c-terminal acidic tail with no known function8,9. Yeast RanGAP, which is a cytoplasmic protein, contains no other known functional domains10. Animal RanGAP, on the other hand, is tethered to the nuclear envelope via interactions with nuclear pore protein NUP358 through a SUMOylated c-terminal domain10. Arabidopsis RanGAP contains an N-terminal domain known as the WPP domain which contains a conserved tryptophan-proline-proline motif and is necessary and sufficient for localization of RanGAP to the nuclear periphery10,11. This localization is accomplished through protein-protein interactions with two families of outer nuclear envelope proteins: the WIPs (WPP Domain Interacting Proteins) and WITs (WPP Interacting Tail-Anchored Proteins)12,13. The WPP domain also targets RanGAP to specific mitotic sites, including the preprophase band, the cortical division site, the cell plate, kinetochores, the spindle midzone, and the outward-growing rim of the phragmoplast7,10,11,14. Arabidopsis thaliana contains two paralogous copies of RanGAP: AtRanGAP1 and AtRanGAP2 which share around 60% amino acid identity with each other and around 20% identity with Saccharomyces cerevisiae Rna1p and Homo sapiens RanGAP10. Although single null mutants of either AtRanGAP1 (AT3G63130) or AtRanGAP2 (AT2G34150) create no observable phenotype, a double null mutant was reported to be female gametophyte lethal and to exhibit arrested nuclear division, indicating a redundant and essential role for the proteins in mitotic progression in plant female gametophytes15. SILK Mutant A mutant called short silique knockdown (SILK) was generated by crossing to obtain a homozygous null of RanGAP1 (rg1-1 allele) and a homozygous knockdown of RanGAP2 (rg2-2 allele) (rg1-1/rg1-1 rg2-2/rg2-2). This line was seen to have a slight 3 developmental delay and shortened siliques. We utilized the silique length phenotype to investigate the role of Arabidopsis RanGAP in a specific part of the plant’s development. Fruit development in Arabidopsis Arabidopsis fruits, called siliques, develop from two fused carpels that form the flower’s gynoecium16,17. Mature fruits are elongated structures with three distinct regions: The replum, which extends longitudinally through the center of the silique and to which the seeds are tethered via their funiculi; the valve, which forms a wall around the seeds; and the valve margins, which separate the valve from the replum at two locations16–20. Although much is known about flower morphogenesis in Arabidopsis, relatively little is known about the further development of flowers into siliques. It is known that elongation of Arabidopsis siliques is initiated after fertilization and occurs in order to make room for the expanding seeds 21,22. This timing is regulated by proteins that actively suppress fruit development until fertilization has occurred21,22. Mutants which initiate silique elongation without fertilization, including fis1, fis2, and fwf, demonstrate the complex regulations that occur to ensure that fruit development and seed development coincide2122. Other genes involved in the development of siliques determine cell fate in the context of the regions outside the seeds once fertilization has released fruit elongation from its inhibition. After fertilization, cells in the valve region divide primarily anticlinaly and differentiate into valve cell types, which include fully developed stomata17. The MADS- Box gene FRUITFULL has been found to be necessary for establishing valve identity, and fruitfull mutants develop short, compact fruits in which the valve region has failed to elongate and differentiate, but which contain a full set of developed seeds16,17. FRUITFULL negatively regulates the valve margin identity gene INDEHICENT which works together with the SHATTERPROOF genes and ALCATRAZ to develop the valve margin region, which is composed of small, thin, lignified cells and which is necessary for the separation of the valves from the replum during seed dispersal18,20. The replum region requires the activity of REPLUMLESS, which negatively regulates both SHATTERPROOF and FRUITFULL in the replum region to prevent valve or valve margin fates from being adopted in the replum, although it is not required for replum fate19. Because so many genes are involved in fruit development, determining which 4 processes are disrupted in the SILK mutant requires a number of phenotypic analyses, the beginning of which will be presented in this thesis. Results: Assays In order to determine whether the GTPase activation function and/or localization of RanGAP are necessary for its role in silique development, we used previously created constructs that contain point mutations in one or both of RanGAP’s functional domains. The first mutant, AAP, includes two point mutations converting tryptophan 18 and proline 19 of RanGAP1 to alanines. This prevents RanGAP’s interaction with its nuclear envelope binding partners WIP and WIT, causing delocalization to the cytoplasm10–13. The two mutants that nullify RanGAP’s GAP activity, termed the “no GAP” mutants, are D330A and N219A. D330A and N219A carry point mutations in aspartate 330 and asparagine 219, respectively, which convert these conserved residues in loop regions of the LRR domain to alanines. These mutations allow the protein to localize properly, but not to interact with Ran8. An additional mutant combines the AAP mutation with the N219A mutation, to create AAP+N219A mutants. SILK plants were transformed with these constructs and the progeny resulting from two generations of selection (T2) were grown alongside wildtype and SILK plants, and their silique lengths were quantified. To determine which part of the developmental process was being affected, a seed count assay was performed as well as sectioning of siliques to observe non-seed cell types. These data were compared and used for analysis of the role of RanGAP in silique development. Silique Length The siliques of SILK plants compared to wildtype were shorter (Figure 2A), with a median at approximately 0.7 centimeters compared to approximately 1.1 centimeters for wildtype. The insertion of wildtype RanGAP1 or AAP into the SILK background